Placental pathology predicts infantile neurodevelopment

The aim of present study was to investigate the association of placental pathological findings with infantile neurodevelopment during the early 40 months of life. 258 singleton infants were enrolled in the Hamamatsu Birth Cohort for Mothers and Children (HBC Study) whose placentas were saved in our pathological division. To assess the infantile neurodevelopment, we used Mullen Scales of Early Learning (gross motor, visual reception, fine motor, receptive language, expressive language) at 10, 14, 18, 24, 32, and 40 months. For obtaining placental blocks, we carried out random sampling and assessed eleven pathological findings using mixed modeling identified ‘Accelerated villous maturation’, ‘Maternal vascular malperfusion’, and ‘Delayed villous maturation’ as significant predictors of the relatively lower MSEL composite scores in the neurodevelopmental milestones by Mullen Scales of Early Learning. On the other hand, ‘Avascular villi’, ‘Thrombosis or Intramural fibrin deposition’, ‘Fetal vascular malperfusion’, and ‘Fetal inflammatory response’ were significant predictors of the relatively higher MSEL composite scores in the neurodevelopmental milestones by Mullen Scales of Early Learning. In conclusion, the present study is the first to report that some placental pathological findings are bidirectionally associated with the progression of infantile neurodevelopment during 10–40 months of age.


Scientific Reports
| (2022) 12:2578 | https://doi.org/10.1038/s41598-022-06300-w www.nature.com/scientificreports/ the outcomes of newborns in cases of severe intrauterine infections, preterm labor, and fetal hypoxia [21][22][23][24] ; however, to the best of our knowledge, it currently remains unclear whether the placental pathology is a predictor of long-term outcomes in offspring, i.e. health and risks of diseases, in the general population. Therefore, we hypothesized that the characteristics of the placental pathology are related to infantile physical and neuronal development in the Japanese population. The Hamamatsu Birth Cohort for Mothers and Children (HBC Study) was designed to elucidate the early developmental trajectories of children living in the community in Japan 25,26 . We recently performed a retrospective analysis of HBC study data and, among the placental pathological findings examined (Fig. 1, Tables 1, 2), identified 'Maternal vascular malperfusion' (Fig. 1A,B) as a significant predictor of a lower body weight and 'Deciduitis' (Fig. 1I) as a significant predictor of a small ponderal index, i.e. lean tendency, throughout the first 18 months of life using 258 whole placentas from singleton pregnancies, which were stored in our pathological division, among 1,258 pregnant women who were enrolled in the HBC study 27 . We planned the present study as a newly evolving investigation of the previous study and conducted a comprehensive analysis to identify the relationships between infantile neurodevelopment during 10-40 months of age, Mullen Scales of Early Learning (MSEL) 28 , and placental pathological findings using the same 258 whole placentas (Table1, 2) from singleton pregnancies in the HBC study.

Results
Enrolled parturients and placentas. Table 1 summarizes the number of pathological findings detected in the placentas examined. Tables 2A,B summarizes the perinatal backgrounds of the participating mothers, fathers, and infants. Their association with infantile neurodevelopment was summarized in Supplemental Table 2 and Table 3. Table 2C summarizes placental measurements. Figure 1 shows the representative placental pathological findings examined.
MSEL scores of five subscales. The MSEL composite scores were cellulated by the summation of net data of all five subscales, i.e. gross motor, visual reception, fine motor, receptive language, and expressive language 28 , at the ages of 10, 14, 18, 24, 32, and 40 months are described in Supplemental Tables.   Fig. 2). Therefore, ' Accelerated villous maturation' , 'Delayed villous maturation' , and 'Maternal vascular malperfusion' were identified as significant predictors of the relatively lower MSEL composite scores in the infantile neurodevelopmental milestones during 10 to 40 months of age. There were no interactions between any of the above placental pathologies and neurodevelopmental outcomes with respect to age in months.
Placental pathology predictive of the relatively higher MSEL composite scores in the infantile neurodevelopmental milestones. A mixed model analysis adjusted for potential confounders showed that MSEL composite scores were significantly higher in infants with positive placental pathological findings of 'Thrombosis or Intramural fibrin deposition' , ' Avascular villi' , 'Fetal vascular malperfusion' , and Fetal inflammatory response' than in those with negative placental pathological findings (Table 3, Fig. 3). Therefore, 'Thrombosis or Intramural fibrin deposition' , ' Avascular villi' , 'Fetal inflammatory response' , and 'Fetal vascular malperfusion' were identified as significant predictors of the relatively higher MSEL composite scores in the infantile neurodevelopmental milestones during 10 to 40 months of age. There were no interactions between any of the above placental pathologies and neurodevelopmental outcomes with respect to age in months.
Placental pathology with no relationship with infantile neurodevelopmental milestones. A mixed model analysis adjusted for potential confounders revealed that MSEL composite scores were similar in infants with positive placental pathological findings of 'Decidual arteriopathy' , 'Maternal inflammatory response' , 'VUE' , and 'Deciduitis' and in those with negative placental pathological findings (Table 3).

Discussion
The present results demonstrated that some pathological findings of the placenta were specifically connected to the achievement of the milestones of infantile neurodevelopment, in a bidirectional manner; i.e. relatively delayed and lower and higher MSEL composite scores (  Fig. 2).
' Accelerated villous maturation' is an abnormal villous branching pattern that is diffusely observed in the placenta, and is suggested to be associated with hypoxic conditions in utero 18,29,30 . Previous studies indicated the strong suppressive effects of fetal hypoxia on physical development after birth in cases of severe fetal growth restrictions. The present study is the first to show that possible hypoxic conditions in utero represented by ' Accelerated villous maturation' are a predictor for the relatively lower scores of MSEL during 10 to 40 months of age (Table 3, Fig. 2A). ' Accelerated villous maturation' is a representative placental pathological finding in the recent concept of 'Maternal vascular malperfusion' by the Amsterdam Placental Workshop Group Consensus Statement 31 , which was also identified as a significant predictor of the relatively lower MSEL composite scores in the neurodevelopmental milestones during 10 to 40 months of age (Table 3, Fig. 2B).
We previously reported that the placental pathological finding of 'Maternal vascular malperfusion' was a predictor for a relatively light body weight during the first eighteen months of life 18 . Therefore, 'Maternal vascular malperfusion' predicts a relatively small body weight 18 as well as the lower MSEL composite scores in the milestones of neurodevelopment during the infantile period. A potentially low supply of maternal blood into the placental intervillous space represented by the pathological finding of 'Maternal vascular malperfusion' may be causatively associated with suspected hypoxic conditions in fetal blood, which may contribute, at least partly, to the programming of a light body weight as well as relatively slow neurodevelopment during the early infantile  32 . However, the pH of the umbilical arteries at delivery, representing acute changes during parturition, was not always associated with the detection of the chronic hypoxic findings of 'Maternal vascular malperfusion' . Therefore, further physiological and epigenetic research is needed to prove this speculation. The pathological finding of 'Delayed villous maturation' was also a predictor of the relatively lower MSEL composite scores in the infantile neurodevelopmental milestones during 10 to 40 months of age (Table 3, Fig. 2C). We cannot currently explain this relationship. However, the placental pathology of 'Delayed villous maturation' has frequently been reported in cases of maternal diabetic complications 9,17,31,33,34 ; therefore, we speculate that chronic high glucose levels, clinical or subclinical, may have programmed the relatively lower MSEL composite scores in the neurodevelopmental milestones during the infantile period. A more detailed investigation is needed to prove this speculation.
In contrast, the pathological findings of 'Thrombosis or Intramural fibrin deposition' , ' Avascular villi' , 'Fetal vascular malperfusion' , and 'Fetal inflammatory response' were significant predictors of the relatively higher MSEL composite scores in the infantile neurodevelopmental milestones during 10 to 40 months of age (Table 3, Fig. 3A-C).
The former two pathological findings of 'Thrombosis or Intramural fibrin deposition' and ' Avascular villi' indicate a localized and insufficient fetal blood supply to specific villus branches. 'Thrombosis or Intramural fibrin deposition' was observed inside the walls of large villus vessels and was regarded as an occlusion and/or the stenosis of upstream fetal blood flow in the specific villous area 9,17,31,34,35 . On the other hand, ' Avascular villi' was histologically defined as the total loss of villous capillaries and was regarded as an insufficiency of downstream fetal blood flow in the specific villous area 9,17,31,34,35 . Therefore, 'Thrombosis or Intramural fibrin deposition' and ' Avascular villi' both indicate the identical trajectory of a decreased fetal blood supply to specific and localized villi. 'Thrombosis or Intramural fibrin deposition' and ' Avascular villi' are representative placental pathological findings in the recent concept of 'Fetal vascular malperfusion' by the Amsterdam Placental Workshop Group Consensus Statement 31 .
Insufficient fetal blood supply to specific villous areas, i.e. 'Thrombosis or Intramural fibrin deposition' , ' Avascular villi' , and 'Fetal vascular malperfusion' , were identified as significant predictors of the relatively higher MSEL composite scores in the infantile neurodevelopmental milestones during 10 to 40 months of age (Table 3, Fig. 3). The pathological finding of 'Fetal inflammatory response' was also a predictor of the relatively higher MSEL composite scores in the infantile neurodevelopmental milestones during 10 to 40 months of age (Table 3, Fig. 3D). It is paradoxical that pathological findings of an insufficient fetal blood supply to specific villous areas and fetal inflammatory response were corelated with relatively higher MSEL composite scores in the infantile neurodevelopmental milestones. We have no clear explanation and cannot fully deny the possible effect of unmeasured confounders. More detailed investigation is necessary.  www.nature.com/scientificreports/ The DOHaD concept highlights improvements in and/or interventions for perinatal, neonatal, and infantile care for the establishment of 'preemptive medicine' against the rapid spread of adult and senile NCDs 4, 36 . It has been proposed as a central strategy of 'preemptive medicine' to identify high-risk individuals in early life as a promising target for earlier interventions 3,37,38 . Extensive efforts have been made over the past few decades to establish effective biomarkers through the application of 'omics' technologies, which may identify individuals at high risk of developing NCDs; however, only a very small number have been translated into routine health care support 39,40 . The present study revealed that some placental pathological findings were associated with changes in infantile neurodevelopment, in addition to our previous findings on body weight and body composition 27 in the Japanese population, suggesting that placental pathological findings are applicable as a type of biomarker for predicting neuronal as well as physical development after birth. Follow-up investigations of the offspring of the present participants are now ongoing. However, more large-scale prospective cohort studies are needed.
There are some limitations in the present study. First of all, present sample size was small and we cannot deny the possibility that future large-scale study may lead to different associations from present ones, between placental pathology and progression of infantile neurodevelopment. We used the criteria of the Amsterdam Placental Workshop Group Consensus Statement, which includes subclassification of stage and/or grade 31 . However, we made assessment as positive vs negative for each individual pathological finding, without consideration of stage nor grade. The differential contribution of the stage and/or grade of each pathological finding is a future aim of the study. The diagnosis of 'Delayed villous maturation' and ' Accelerated villous maturation' are somewhat subjective even based on Amsterdam Placental Workshop Group Consensus Statement 31 . Placental pathological screening was carried out only pregnant women who agreed; resultantly, the number of cesarean deliveries was 149 among 258 subjects. Since majority of cesarean indication was elective cesarean section before onset of labor, average birth weight of 2815 g of the enrolled subject was somewhat smaller than Japanese average. The perinatal backgrounds of parents and infants of the rerolled subjects, who agreed to store the placentas, were not exactly identical to the unenrolled ones (Table 2). We enrolled only singleton pregnancies; while unenrolled group included twin progenies, which is one of the different backgrounds of two groups. Other difference in enrolled placentas were higher maternal age, higher maternal BMI, higher paternal age, higher incidence of Diabetes mellitus, higher incidence of postpartum maternal depression, lower birth weight, lower gestational age at birth, and lower umbilical arterial pH, than those in not enrolled ones, respectively (Table 2A). It is undeniable that In conclusion, the present study is the first to report that some placental pathological findings are bidirectionally associated with the progression of infantile neurodevelopment during 10-40 months of age.

Methods
Subjects. The present study was conducted as part of an ongoing cohort study (the HBC Study), which has been described elsewhere 25,26 . We consecutively contacted all pregnant women (n = 1,258) who were expected to give birth at our two research sites, the Hamamatsu University Hospital and Kato Maternity Clinic, which are both situated in Hamamatsu city, and who gave birth between 20 December 2007 and 31 October 2011. We previously confirmed that the enrolled parturients were representative of Japanese parturients in terms of age, socioeconomic status, parity, and the birthweight and gestational age of the child 25,26 . Among 1,258 subjects, we initially analyzed 261 whole placentas from singleton pregnancies because the parents had agreed to store their whole placentas in our pathological division. However, we excluded three placentas from the analysis: one infant had died, another had a confirmed diagnosis of Down's syndrome with severe congenital heart disease, and the parents of the remaining infant refused to participate in this study after delivery. The remaining 258 (98.9%) placentas were analyzed.
All participating parturients were given a complete description of the present study, and provided written informed consent to participate. They were followed from entry into the study during mid-pregnancy to 40 months after childbirth.
Preparation of placental tissue blocks. After weighing and an examination of gross morphology, whole placentas were stored in our pathological division after being vacuum-sealed in plastic packages with 10% formaldehyde (0.1 M sodium citrate buffer, pH 7.4). Seven paraffin blocks were systematically obtained from each placenta for the pathological examination by systematic random sampling, as previously described 18,19,27 . In brief, 5-mm-wide linear parallel slices of placental tissue were cut at an interval of approximately 3 cm perpendicular to the greatest dimension of the placental axis. All linear slices were vertically cut into small pieces at an interval of 3 cm. Seven blocks per placenta were obtained from seven randomly selected pieces of the placental parenchymal tissue obtained. Each block was made vertically from the maternal side to the fetal side. Two rolls of extraplacental membranes per placenta were together embedded in a block to make a single section. Each block was cut into 3-µm-thick sections and then subjected to hematoxylin and eosin (HE) staining. Eight sections (1) ' Accelerated villous maturation' was diagnosed as increased numbers of placental villi with the focal formation of tight adherent villous clusters 9,17,31,34 typically with syncytial knots, increased perivillous fibrin, and the distal villous hypoplasia of small terminal villi 43 (Fig. 1A), (2) 'Decidual arteriopathy' was diagnosed as vascular lesions including the fibrinoid necrosis of decidual vessels or arthrosis at the basal plate 9,31,34,44 (Fig. 1B), (3) 'Thrombosis or Intramural fibrin deposition' was diagnosed as localized, protuberant mural lesions composed of proliferating fibroblasts intermixed with fibrin and erythrocytes in the walls of large placental vessels according to the description by Desa 9,17,31,34,35 (Fig. 1C), (4) ' Avascular villi' was diagnosed as the total loss of villous capillaries and bland hyaline fibrosis in an older lesion 31 (Fig. 1D), (5) 'Delayed villous maturation' was diagnosed as a monotonous villous population with reduced numbers of vasculosyncytial membranes, increases in the size of distal villi, increased numbers of stromal cells, and interstitial fluid uniformly distributed throughout the villous stroma 9,17,31,33,34 (Fig. 1E), (6) 'Maternal inflammatory response' was diagnosed by the infiltration of neutrophils into the connective tissues of the chorionic plate and/or amnion basement membrane in the fetal surface of the placenta 9,17,31,34,45 (Fig. 1F), (7) 'Fetal inflammatory response' was diagnosed by the infiltration of neutrophils into umbilical vessels or chorionic plate vessels 9,17,31,44 (Fig. 1G), (8) 'VUE' was diagnosed by lymphohistiocytic inflammation predominantly localized to the villous stroma of terminal villi despite the absence of clinical symptoms of apparent infection in mothers or infants 9,17,31,34,46 (Fig. 1H), and (9) 'Deciduitis' was employed as one of the findings of 'others' , following the criteria of the Amsterdam Placental Workshop Group Consensus Statement 31 , which was diagnosed by the criteria of the presence of ≥ 50 lymphocytes/per high-power field 47 , often accompanied by chronic villitis or decidual necrosis 9,44 (Fig. 1I). 'Maternal vascular malperfusion' and 'Fetal vascular malperfusion' were diagnosed inconsideration of the Amsterdam Placental Workshop Group Statement 31 . Maternal vascular a vascular malperfusion was diagnosed if ' Accelerated villous maturation' and/ or 'Decidual arteriopathy' were observed. Fetal vascular malperfusion was diagnosed in case 'Thrombosis or Intramural fibrin deposition' and/or ' Avascular villi' were observed. In the present study, each of the eleven pathological findings were assessed as positive or negative through the majority decision of independent and blind surveys conducted by three researchers, i.e. Drs. Chizuko Yaguchi, Naomi Furuta, and Yoshimasa Horikoshi, as previously described 27 . They are researchers of placental pathology 18,27,48,49 . Assessment of infantile neurodevelopment. We used MSEL 28 to evaluate neurodevelopment in infants. MSEL is a composite scale for assessing child development and comprises five subscales: gross motor, visual reception, fine motor, receptive language, and expressive language 28 . Measurements were performed when infants reached the ages of 10, 14, 18, 24, 32, and 40 months. Prior to follow-up assessments of the birth cohort, two experienced clinicians completed 3-month video training sessions, through which the agreement of their scoring of each item on the MSEL scale was attained. Separate 3-month video training sessions were set up and included an additional five assessors (child health professionals) who engaged in actual assessments. Developmental assessments with MSEL were conducted by referring to previously established data 28 . To evaluate the developmental trajectories of each of the five domains, MSEL-T scores, which are equivalent to Z-scores, but with a mean of 50 and standard deviation of 10, were generated.
Information on the demographic characteristics of mothers was collected during the pregnancy of enrolled parturients and included the age of the mother, parity, smoking, and pre-pregnancy height and weight 25,26 . Perinatal variables were collected from medical records 25,26 . Statistical analysis. MSEL composite scores 28 at 10, 14, 18, 24, 32, and 40 months were set as dependent variables, and pathological findings as the independent variable. Continuous variables were reported as the mean ± SD. In comparisons of MSEL composite scores between two groups in the initial assessment (positive vs negative for each individual pathological finding), we performed the Student's t-test or Mann-Whitney U test where appropriate. Significance was set at a p value of 0.05.
To assess the longitudinal trajectories of MSEL composite scores 28 , we adopted the method of mixed modeling 27 using the mixed command provided by the generic statistical software, Stata version 16.1. Mixed modeling has the strength of analyzing longitudinal patterns of development in association with the fixed effect of the placental pathology that occurred long before anthropological measurements took place after birth 27 . Furthermore, mixed modeling allowed us to incorporate all available data into the analysis 27 even if some data had missing values in the repeated measurements of MSEL composite scores. A growth curve model, i.e. mixed modeling with a random intercept and random slope 27 , was built for MSEL composite scores, during 10 to 40 months of age, adjusted for age in months at the time of infant assessments. We then incorporated all available covariates deemed to be potential confounders into the above analysis. Covariates included in the analysis were maternal parity, gestational weeks, and infantile sex, which may be related to placental pathological findings and MSEL composite scores 27 . Mixed model analyses of longitudinal data were conducted between 10 and 40 months in order to identify differential effects on MSEL composite scores between the presence and absence of several pathological findings. We subsequently entered all available indices of the placental pathology into the above